Cancer vaccines have been developed as a promising way to boost cancer immunity. However, their clinical potency is often limited due to the imprecise delivery of tumor antigens. To overcome this problem, we conjugated an endogenous toll like receptor (TLR)2/6 ligand, UNE-C1, to human papilloma virus type 16 (HPV-16)-derived peptide antigen, E7, and found that the UNE-C1-conjugated cancer vaccine (UCV) showed significantly enhanced antitumor activity in vivo compared to the noncovalent combination of UNE-C1 and E7. The combination of UCV with PD-1 blockades further augmented its therapeutic efficacy. Specifically, the conjugation of UNE-C1 to E7 enhanced its retention in inguinal draining lymph nodes, the specific delivery to dendritic cells and E7 antigen-specific T-cell responses, and antitumor efficacy in vivo compared to the noncovalent combination of the two peptides. These findings suggest the potential of UNE-C1 derived from human cysteinyl-tRNA synthetase 1 (CARS1) as a unique vehicle for the specific delivery of cancer antigens to antigen-presenting cells via TLR2/6 for the improvement of cancer vaccines.